[79] Resolution and Reconstitution of ATP Synthesis and ATP-Dependent Functions of Liver Mitochondria

The concentration of urea is critical in preparing urea particles that are reconstitutively active in catalyzing adenosine triphosphate (ATP) synthesis. If 8 M urea is used to treat inner membrane vesicles in place of the 6 or 6.4 M urea, 4 M urea particles are obtained. These particles, in analogy to 3 or 3.2 M urea particles, readily bind purified F₁ ATPase. However, the Fl-restored, 4 M urea particles have <30% maximal capacity to catalyze adenosine triphosphate (ATP) synthesis. F₁-restored, 3 or 3.2 M urea particles, on the other hand, catalyze rates of ATP synthesis that are comparable to rates catalyzed by the starting inner membrane fraction. It is also critical for best results to assay the F₁-restored urea particles immediately after centrifugation and resuspension. Failure to do so within 30 min to 1 hr results in loss of the ATP or AMP-PNP activating effect and suboptimal rates of ATP synthesis is observed.